Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) are single transmembrane proteins which are expressed in a wide spectrum of cells and tissues including immunocompetent cells such as macrophages and neutrophils, vascular endothelial cells, and renal intrinsic cells such as renal tubular epithelial cells (Folia Biol(Praha)., 2005, Vol. 51, No. 6, p. 188-197). TLR2 forms a complex with TLR1 or TLR6, and reacts with bacterial components such as peptidoglycan or lipoprotein. Meanwhile, TLR4 forms a complex with a myeloid differentiation factor 2 (MD2) protein, a CD14 protein, or the like, and reacts with bacterial components such as lipopolysaccharide typified by lipopolysaccharide (LPS). In addition, these receptors are activated by binding to damage associated molecular patterns (DAMPs) which are an endogenous tissue damage factor, and transmit signals into cells. Activation of TLR2 and TLR4 induces the expression of inflammatory cytokines such as tumor necrosis factor α (TNFα) and interleukin 6 (IL-6) and elicits an inflammatory response, for example, through the activation of a nuclear factor κB (NF-κB) which is a transcription factor (Nat. Immunol., 2010, Vol. 11, No. 5, p. 373-384).
Such TLR2- and TLR4-mediated activation of various cells is known to be involved in immune inflammatory diseases such as sepsis, acute renal failure, chronic renal disease, acute respiratory distress syndrome, scleroderma, acute pancreatitis, and chronic obstructive pulmonary disease.
In association with sepsis, it has been reported that genetically TLR2-deficient animals (knockout mice) in a Salmonella infection model which is a sepsis model exhibit a survival rate improvement, TLR4 knockout mice in an Escherichia coli infection model exhibit a survival rate improvement, and mice with a double knockout of TLR2 and TLR4 in each model exhibit a significantly improved survival rate as compared to mice with a single knockout of TLR2 or TLR4 (J. Exp. Med., 2008, Vol. 205, p. 1747-1754). In addition, it has been reported that the survival rate is not improved with administration of an anti-TLR2 antibody (T2.5) or an anti-TLR4 antibody (1A6) alone in the above two models, whereas the survival rate is improved with combined administration of both antibodies (J. Exp. Med., 2008, Vol. 205, p. 1747-1754).
In association with acute pancreatitis, it has been reported that TLR4 knockout mice in a taurocholate-induced pancreatitis model exhibit lowering of blood amylase, pancreatic myeloperoxidase, and pancreatic tissue damage (Inflamm. Res., 2011, Vol. 60, p. 1093-1098). Further, it has been reported that the expression level of TLR2 and TLR4 in the pancreas is increased in a caerulein-induced pancreatitis model (Pancreas, 2013, Vol. 42, p. 114-122).
As an antibody binding to human TLR2, a mouse monoclonal antibody T2.5 (Patent Document 1), a mouse monoclonal antibody 11G7 (Patent Document 2), and a humanized monoclonal antibody OPN305 (Patent Document 3) of T2.5 have been reported. Among them, 11G7 inhibits TLR2/TLR1 signals but does not inhibit TLR2/TLR6 signals, whereas OPN305 has been reported to inhibit both signals. Specifically, it has been reported that in experiments using a THP-1 cell which is a human monocytic cell, OPN305 has a neutralizing activity against both Pam3CSK4 (TLR2/TLR1 agonist) stimulation and FSL-1 (TLR2/TLR6 agonist) stimulation, and inhibits the elevation of a mouse serum inflammatory cytokine concentration induced by administration of Pam3CSK4 (Patent Document 3).
As an antibody binding to human TLR4, a mouse monoclonal antibody HTA125 (Non-Patent Document 1), mouse monoclonal antibodies 18H10, 16G7, 15C1 and 7E3 (Patent Document 4), a humanized monoclonal antibody hu15C1 of 15C1 (Patent Document 5), and humanized monoclonal antibodies 1A6 and 1E11.C2E3 and the like discovered by random mutations to the complementarity determining region of hu15C1 (Patent Document 6) have been reported. Among them, with respect to mouse monoclonal antibodies, 15C1 has been shown to have the highest neutralizing activity from the experimental results using human blood (Patent Document 4), and the humanized monoclonal antibody hu15C1 of 15C1 has been confirmed to have a neutralizing activity by experiments using the same human blood (Patent Document 5). Further, the humanized monoclonal antibody 1E11.C2E3 has been reported to have a superior neutralizing activity than hu15C1 in experiments using human blood (Patent Document 6).
In addition, it has been reported that, in an IL-6 production evaluation system for stimulation of Escherichia coli, Pseudomonas and Klebsiella subjected to a heat treatment using human blood, the anti-human TLR2 antibody (T2.5) weakly inhibits IL-6 production and the anti-human TLR4 antibody (15C1) inhibits about 50% of IL-6 production, whereas the combined addition of both antibodies almost completely inhibits IL-6 production (Patent Document 7).
However, a bispecific antibody binding to human TLR2 and human TLR4 has not been reported thus far.